What is the difference between suspension and resuspension buffers ?
I am trying to more completely understand what I am doing in my lab protocol entitled “Cloning of export signals by in vivo gene fusions using bla beta lactamase as a reporter gene”. What are suspension and resuspension buffers, and what are they doing in the context of such an experiment?
Also If someone could tell me what exactly is meant by “plasmid purification” that would be great. Does that mean you are extracting the non-vector “natural” plasmids from the ecoli, or other bacteria, so that you can then cut them with restriction enzymes? I understand the premise of the experiment, I think, but why we do all the procedural things is confusing.
I guess you are probably using some kind of kit to do this, with numbered buffers? All that those terms mean in this context is to differentiate between different buffers that do different things. For example, when you spin down the cells to remove the growth medium, then you suspend the pellet in one buffer – the suspension buffer – but then depending on what you are doing you may need to spin them down again and resuspend the pellets in another buffer – the resuspension buffer. They may also be called buffer 1, 2, 3 etc.
“Plasmid purification” is usually used when you have already transformed your desired construct into your vector, and you wish to extract it again. This may sound counter-productive, but when you do the whole process from scratch – design & order primers, run PCR, run DNA cleanup – you want to make sure you have the correct product before you transform it into your bacteria and do a large-scale prep. To do this, you can’t just sequence the PCR product, because it is not sufficiently concentrated, and it may be a mixture of correct & incorrect products. Instead, you transform the PCR product into a cloning strain such as MC1061, and grow up with antibiotic selection. This allows you to provisionally select a colony which shows the correct antibiotic resistance, perform a plasmid extraction & send the plasmid for sequencing. If it shows the correct construct, the extracted plasmid can be stored at -20oC for future transformations
Agricultural Reporter – Working in Canada
